Targeted modified TNF family members

ABSTRACT

The present invention relates to a modified cytokine of the TNF superfamily, with reduced activity to its receptor, wherein said modified cytokine is specifically delivered to target cells. Preferably, said modified cytokine is a single chain variant of the TNF superfamily, even more preferably, one or more of the chains carry one or more mutations, resulting in a low affinity to the receptor, wherein said mutant cytokine is specifically delivered to target cells. The targeting is realized by fusion of the modified cytokine of the TNF superfamily to a targeting moiety, preferably an antibody or antibody-like molecule. The invention relates further to the use of such targeted modified cytokine of the TNF superfamily to treat diseases.

The present invention relates to a modified cytokine of the TNFsuperfamily, with reduced activity to its receptor, wherein saidmodified cytokine is specifically delivered to target cells. Preferably,said modified cytokine is a single chain variant of a member of the TNFsuperfamily, even more preferably, one or more of the chains carry oneor more mutations, resulting in a low affinity to the receptor, whereinsaid mutant cytokine is specifically delivered to target cells. Thetargeting is realized by fusion of the modified cytokine of the TNFsuperfamily to a targeting moiety, preferably an antibody orantibody-like molecule. The invention relates further to the use of suchtargeted modified cytokine of the TNF superfamily to treat diseases.

The TNF superfamily consists of pro-inflammatory cytokines with crucialfunctions in the immune system by regulating cell death, proliferationand differentiation. In addition, members of the family were describedto exert functions on bone metabolism, the nervous system, onneo-vasculature and carcinogenesis. It contains 19 ligands, type II(intracellular N terminus and extracellular C terminus) transmembraneproteins, which are biologically active as self-assembling, non-covalentbound homotrimers. Although most TNF superfamily ligands are synthesizedas membrane-bound proteins, soluble forms can be generated byproteolytic cleavage. All of them bind to one or more molecules from theTNF receptor superfamily through their C-terminal TNF homology domain,which exhibits ˜20-30% sequence homology between family members. So far,29 TNF superfamily receptors have been identified in humans. These areprimarily type I (extracellular N terminus, intracellular C terminus)transmembrane glycoproteins with a cystein-rich motif in theligand-binding extracellular domain. However, there are some exceptionslike TRAIL-R3 that is attached to the membrane by a covalently linkedC-terminal glycolipid. Soluble receptors can be generated by proteolyticcleavage (e.g. TNF-R1 and TNF-R2) or by alternative splicing of the exonencoding the transmembrane domain. The receptors of this superfamily canbe divided in 3 groups based on their signaling properties: receptorswith a cytoplasmic death domain that induce apoptosis; receptors with aTRAF-interacting motif that induce several signaling pathways such asNF-κB, JNK, p38, ERK and PI3K; and the decoy receptors that lackintracellular signaling domains. TNF induces apoptosis throughinteraction with TNF-R1 (p55), while binding to TNF-R2 (p75, primarilyexpressed on immune cells) promotes proliferation. TRAIL signaling ismore complex as it can bind to two death receptors (TRAIL-R1 (DR4) andTRAIL-R2 (DR5)), to two decoy receptors (TRAIL-R3 (DCR1) and TRAIL-R4(DCR2)) and to the soluble osteoprotegerin (OPG). Binding to one of thelatter three receptors inhibits TRAIL-mediated apoptosis as it tethersTRAIL away from the death receptors (Gaur and Aggerwal, 2003; Hehlgansand Pfeffer, 2005; Huang and Sheikh, 2007).

The death-inducing TNF superfamily members TNF, CD95L (FasL) and TRAILare potential therapeutics for cancers that express their respectivereceptor TNF-R1, CD95, TRAIL-R1 and TRAIL-R2. In fact, TNF wasoriginally discovered more than 25 years ago as a factor withextraordinary antitumor activity, by causing hemorrhagic necrosis ofcertain tumors in vivo. Later it became clear that the selective damageattributed by TNF to tumor neovasculature also defines its anti-tumorpotential (Lejeune et al., 2006; van Horssen et al., 2006).Unfortunately, systemic use of TNF in cancer treatment is still hamperedby its shock-inducing properties. It is currently only clinically usedin the setting of isolated limb perfusion in combination withchemotherapy to treat soft tissue sarcomas and in-transit melanoma(Roberts et al., 2011). Also CD95L is toxic when administeredsystemically as it causes lethal hepatotoxicity due to massivehepatocyte apoptosis (Galle et al., 1995). TRAIL, however, has beenshown to induce apoptosis in cancer cells with little or no cytotoxicityagainst non-transformed cells, and clinical trials in various advancedcancers report stable disease in many cases. Still, to obtain sufficientoverall therapeutic activity combined treatment is required, whichimplies possible side effects due to sensitization of normal cells toTRAIL-induced apoptosis (Ashkenazi and Herbst, 2008; Falschlehner etal., 2009). Different approaches have been undertaken to minimize thetoxicity upon systemic administration of death-inducing TNF superfamilymembers, such as mutant TNF with lower toxicity and higher efficiency(Li et al., 2012), delivery of TNF or TRAIL, normally as a single chainconstruct, by tumor-specific moieties (de Bruyn et al., 2013; Gregorc etal., 2009; Liu et al., 2006; Siegemund et al., 2012; Wang et al., 2006),chimeric soluble CD95L (Daburon et al., 2013) or agonistic TRAIL-R1-,TRAIL-R2 or CD95-specific antibodies (Johnstone et al., 2008; Ogasawaraet al., 1993; Fox et al., 2010). Some of them can increase thetherapeutic index but never to such an extent that it dramaticallyimproves clinical outcome.

Surprisingly, we found that it is possible to make a constructcomprising a cytokine of the TNF superfamily, wherein the cytokine ismodified to lower the affinity towards the receptor, wherein saidcytokine is linked to a targeting moiety, and wherein said construct hasa strongly reduced systemic toxicity, and only shows significantbiological activity towards the cells that are targeted by the targetingmoiety.

A first aspect of the invention is a construct, comprising (i) threecopies of a cytokine chain of the TNF superfamily, wherein the resultingcytokine is modified (referred to as “modified cytokine”) so that theaffinity towards its receptor is lowered, (ii) a linker sequence and(iii) a targeting moiety, wherein said linker sequence is linking thecytokine copies to the targeting moiety. A construct, as used here, canbe any proteinaceous construct known to the person skilled in the art,including, but not limited to chemically modified proteins, proteincomplexes and fusion proteins. In one preferred embodiment, individual,self-trimerizing cytokine chains are used, wherein one or more of thechains may be linked to the targeting moiety. In another preferredembodiment, the three copies are presented as a single chain cytokine;in a preferred embodiment, the copies are separated by a linker sequenceto facilitate the presentation of the cytokine in a trimeric form. It isclear for the person skilled in the art that mixed forms, with one freecytokine chain and two cytokine copies linked to each other, are alsopossible. The resulting trimeric cytokine carries a modification thatlowers its biological activity, compared to the wild type cytokine. Sucha modification can be a modification that decreases the activity of thenormal wild type cytokine, or it can be a modification that increasesthe activity of a homologous, non-endogenous TNF family cytokine (suchas, but not limited to, a TNF family cytokine of another species that isnot binding to a human TNF family cytokine receptor). Modifications canbe any modification reducing or increasing the activity, known to theperson skilled in the art, including but not limited to chemical and/orenzymatic modifications such as pegylation and glycosylation, fusion toother proteins, and mutations. In case two or more copies of thecytokine are presented as a single chain, the length of the linker maybe adapted to disturb the normal trimeric structure, resulting in alower activity toward the receptor. Alternatively, special amino acidsmay be incorporated in the linker to modify the structure; said aminoacids may further be modified. As a non-limiting example, a lysine maybe incorporated in the linker to allow pegylation. Preferably saidmodification is a mutation, even more preferably it is a mutationdecreasing the affinity of cytokine towards its receptor. A reducedaffinity and a consequent reduced biological activity as used here meansthat the modified cytokine has a biological activity of less than 70% ofthe biological activity of the wild type cytokine, even more preferablyless than 60% of the biological activity of wild type cytokine, morepreferably less than 50% of the biological activity of the wild typecytokine, more preferably less than 40% of the biological activity ofwild type cytokine, more preferably less than 30% of the biologicalactivity of the wild type cytokine, more preferably less than 20% of thebiological activity of the wild type cytokine, most preferably less than10% of the biological activity of the wild type cytokine as compared towild type cytokine that normally binds to the receptor. Preferably, themodified cytokine of the TNF superfamily is a mutant of the wild typecytokine of the TNF superfamily and the activity is compared with thewild type cytokine of the TNF superfamily. The affinity and/or theactivity can be measured by any method known to the person skilled inthe art. The affinity of the mutant TNF to the receptor, in comparisonto the affinity of the wild type TNF to the receptor can be measured byScatchard plot analysis and computer-fitting of binding data (e.g.Scatchard, 1949) or by reflectometric interference spectroscopy underflow through conditions, as described by Brecht et al. (1993).

Preferably, said cytokine is selected from the group, consisting ofFasL, TRAIL, TNF, CD30L, CD40L, OX40L, RANKL, TWEAKL, LTalpha, LTbeta,LIGHT, CD27L, 41BBL, GITRL, APRIL, EDA, VEGI, and BAFF. Preferably, saidcytokine is presented as a single chain, wherein the chains areconnected by a linker sequence.

The modified cytokine is linked to a targeting moiety. “Linked” as usedhere may be by a covalent binding, or it may be by an affinity binding.In a non-limiting example, said targeting moiety may be a bispecificantibody, directed to a binding site on the target cell for onespecificity, and to the modified cytokine, or to a tag fused to saidcytokine for the other specificity. In another non-limiting example, thetargeting moiety may be chemically linked to the modified cytokine, orit may be a recombinant fusion protein. Preferably, said targetingconstruct is a recombinant fusion protein. Preferably, said targetingmoiety is targeting the cytokine to a tumor environment, particularly tothe tumor vasculature (e.g. to endothelial cells of the tumor, typicallyneo-endothelial cells). Even more preferably, a targeting moiety is abinding molecule that can direct the fusion protein towards a bindingsite on a cell that is expressing a receptor for the cytokine of the TNFsuperfamily, preferably a receptor capable of interacting with themodified cytokine, by specific interaction between the binding site andthe binding molecule. In one preferred embodiment, said binding moleculeis a small compound, specifically binding to a molecule situated on theoutside of the cell. In another preferred embodiment, said bindingmolecule is a sugar structure, directed towards a lectin-like moleculeexpressed on the cell wall. In another preferred embodiment said bindingmolecule is a peptide, targeting the tumor environment, preferably tothe tumor vasculature. Such peptides are known to the person skilled inthe art, and include, but are not limited to, NGR (targeting CD13isoforms expressed in tumor vessels) and RGD peptides (Yang et al.,2011; WO2005054293). Preferably, said peptide is an RGD-4C peptide (Arapet al., 1998) which targets the α_(v)β₃ integrin. In still anotherpreferred embodiment, said binding molecule is a protein comprising abinding domain. Binding domains are known to the person skilled in theart. Non-limiting examples of such binding domains are carbohydratebinding domains (CBD) (Blake et al, 2006), heavy chain antibodies(hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al.,1994), the variable domain of camelid heavy chain antibodies (VHH), thevariable domain of the new antigen receptors (VNAR), affibodies (Nygrenet al., 2008), alphabodies (WO2010066740), designed ankyrin-repeatdomains (DARPins) (Stumpp et al., 2008), anticalins (Skerra et al.,2008), knottins (Kolmar et al., 2008) and engineered CH2 domains(nanoantibodies; Dimitrov, 2009). Preferably, said targeting moiety is ananobody.

In a preferred embodiment, the targeting moiety is linked to themodified cytokine in a recombinant fusion protein. The targeting moietymay be fused directly to the mutant cytokine, or it may be fused withthe help of a linker fragment. Preferably, said linker is a GGS linker.Even more preferably, said linker consists of at least 5 GGS repeats,more preferably of at least 10 GGS repeats, more preferably of at least15 GGS repeats, more preferably said linker is a linker consisting of17-23 GGS repeats, most preferably said linker consists of 20 GGSrepeats. The targeting moiety may be fused at the amino-terminal or atthe carboxy-terminal end of the mutated cytokine; preferably saidtargeting moiety is fused at the amino-terminal extremity of the mutatedcytokine molecule. Apart from the mutant cytokine and the targetingmoiety, the construct may further comprise other domains such as, butnot limited to, a tag sequence, a signal sequence, another cytokine oran antibody.

Preferably, the targeting moiety is directed towards a target selectedfrom the group consisting of CD20, CD33, CD47, CD70, PSCA, PSMA, Her2,c-Met, EGFR, Axl, tenascin C, α_(v)β₃ integrin, fibronectin EDA end EDBdomains, fibronectin type III (FNIII) repeats (A1-D), tenascin-C, andCD13, and tumor cell-specific splice variants thereof. When thetargeting moiety targets to the tumor vasculature, particularlyenvisaged targets include, but are not limited to, CD13, α_(v)β₃integrin, fibronectin EDA end EDB domains, fibronectin type III (FNIII)repeats (A1-D), and tenascin-C. According to alternative particularembodiments, said targeting moiety is directed to CD20. It isparticularly envisaged that the targeting moiety is an antibody (or ananobody), as antibodies against these targets are readily availableand/or can easily be generated.

The mutation in the cytokine chain can be any mutation known to theperson skilled in the art, such as a deletion, an insertion, or a pointmutation. At least one chain has at least one mutation; however, severalmutations may be combined in one chain. The three chains may carry thesame mutations, or different mutations. Preferably said mutations arelowering the affinity of the cytokine to its receptor or one of itsreceptors. Preferably said mutation is a point mutation.

In one preferred embodiment, the cytokine is TNF, and the construct istargeted towards a marker, expressed on a TNFR1 and/or TNFR2 expressingcell. In another preferred embodiment, said marker is a tissue specificmarker, even more preferably said marker is a neo-vasculature tissue orcancer tissue specific marker. Most preferably, said marker is selectedfrom the group consisting of CD20, Her2, c-Met, EGFR, tenascin C,α_(v)β₃ integrin, and CD13.

Preferably, the modified cytokine is a mutant TNF wherein the mutationis selected from the group consisting of mutations on position R32, N34,Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115,E127, N137, D143, A145. Even more preferably, said mutation is selectedfrom the group consisting of TNF R32G, N34G, Q67G, H73G, L75G, L75A,L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S,T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G andA145T. Even more preferably, said mutation is selected from the groupconsisting of Y87X, I97X and Y115X. Most preferably, said mutation isselected from the group consisting of TNF Y87Q, Y87F, I97A, I97S, Y115Aand Y115G (numbering according to the human TNF sequence, genbankaccession number BAG70306, version BAG70306.1 GI: I97692685). Themutation may be present in one, two or all three copies of the trimericTNF. Different copies within the trimeric construct may carry differentmutations; several mutations may be combined in one or more of thechains. Apart from the cited mutations, other mutations may be presentin one or more chains.

Preferred regions for mutation in TRAIL are T127-R132, E144-R149,E155-H161, Y189-Y209, T214-I220, K224-A226, W231, E236-L239, E249-K251,T261-H264 and H270-E271 (Numbering based on the human sequence, genbankaccession number NP_003801, version NP_003801.1, GI: 4507593).

Another aspect of the invention is a fusion protein according to theinvention for use as a medicament. In one preferred embodiment, thefusion protein according to the invention is for use in treatment ofcancer.

This is equivalent as stating that methods of treating cancer in asubject in need thereof are provided, comprising administering a fusionprotein as described herein to said subject. The cancer is therebytreated. This can for instance be evaluated by evaluating tumor size, asshown in the Examples (see also FIG. 16).

Subjects suitable for treatment are typically mammals, most typicallyhumans. However, treatment of non-human animals is also envisagedherein. Examples of non-human animals that can be treated include, butare not limited to, horses, cows, sheep, pigs, goats, cats, dogs, andother domesticated animals. If non-human animals are envisaged fortreatment, it is particularly envisaged that the modified cytokine isfrom the species to be treated. Modifications by mutation are thenmodifications of the residues in homolog positions compared to the humansequence. By way of non-limiting example, as shown in the Examplessection, in mouse TNF, the residue that is a homolog of Y87 in human TNFis at position 86 (Y86). This can be mutated as detailed above (e.g.Y86F or Y86Q).

Different forms of cancer can be treated using this strategy.Essentially, any tumor that can be targeted (directly or indirectly,through the tumor environment) with a targeting moiety, therebyreactivating the modified TNF family cytokine, and thus inducing tumorcell death, is suitable for treatment.

Particularly envisaged cancers thus are those that can be readilytargeted. According to particular embodiments, the targeting is to thetumor vasculature. Accordingly, highly vascularized tumors areparticularly envisaged. Examples of such tumors are those that can betreated with anti-angiogenic approaches, such as anti-VEGF drugs oranti-angiopoietin/Tie2 agents. These include, but are not limited to,breast cancer, renal cell carcinoma, colorectal cancer, non-small celllung cancer (NSCLC), hepatocellular carcinoma, pancreatic cancer,glioblastoma, ovarian cancer, gastric cancer, prostate cancer, melanoma,gastrointestinal stromal tumor (GIST), neuroendocrine tumors, softtissue sarcoma, medullary thyroid cancer, and endometrial cancer (seee.g. Welti et al., 2013, particularly Table 1 and Supplemental Table 1therein).

According to particular embodiments, the cancer is a solid tumor.However, it should be noted that also hematological cancers such asleukemias (e.g. CML, AML), multiple myeloma and lymphomas can be treatedwith anti-angiogenic agents (Schmidt and Carmeliet, 2011; Roccaro etal., 2006). Thus, according to alternative embodiments, the cancer is atumor of the hematopoietic and/or lymphoid tissues (see also Example12).

As angiogenesis plays a major role in tumor metastasis, and inactivating metastatic lestions, according to particular embodiments, thecancer is a metastatic cancer. The increased presence of neo-endothelialcells will make these cancers more susceptible to molecules targeted tomarkers of these cells (the tumor vasculature markers described above).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Representation of the structural elements of the differentset-ups of the sc hTNF-nanobody fusion protein.

FIG. 2: Firefly luciferase activity induced by the indicated sc hTNFpreparations, as compared to WT hTNF, on HekT cells (panel A) or Hek-mLRcells (panel B). Both were transiently transfected with the NF-κBluciferase reporter.

FIG. 3: Firefly luciferase activity induced by the indicated sc hTNFpreparations carrying a linker with 6 GGS repeats on HekT cells (panelA) or Hek-mLR cells (panel B). Both were transiently transfected withthe NF-κB luciferase reporter.

FIG. 4: Firefly luciferase activity induced by the indicated sc hTNFpreparations carrying a linker with 13 GGS repeats on HekT cells (panelA) or Hek-mLR cells (panel B). Both were transiently transfected withthe NF-κB luciferase reporter.

FIG. 5: Firefly luciferase activity induced by the indicated sc hTNFpreparations carrying a linker with 19 GGS repeats on HekT cells (panelA) or Hek-mLR cells (panel B). Both were transiently transfected withthe NF-κB luciferase reporter.

FIG. 6: Fold induction of IL-6 mRNA levels upon treatment of SK-BR-3cells with 500 ng/ml of the indicated sc hTNF preparations compared tothe levels in untreated cells and cells stimulated with the Her2nanobody. Data represents the mean±SD of 2 independent experiments(n=4).

FIG. 7: % activity of hTNF mutants compared to WT hTNF as measured bytoxicity on MCF7 cells (a breast cancer cell line).

FIG. 8: Toxicity on MCF7 (panel A) or MCF7-mLR (panel B) cells oftargeted modified TNFs coupled to mLR NB (NB C-terminally of TNF).

FIG. 9 Toxicity on MCF7 (panel A) or MCF7-hCD20 (panel B) cells oftargeted modified TNFs coupled to hCD20 NB (NB C-terminally of TNF).

FIG. 10: Toxicity on MCF7 (panel A) or MCF7-hCD20 (panel B) cells oftargeted modified TNFs coupled to hCD20 or control Bcll10 NB (NBN-terminally of TNF).

FIG. 11: Toxicity on MCF7 (panel A) or MCF7-hCD20 (panel B) cells oftargeted modified TNFs containing sc hTNF with combined mutations (NBN-terminally of TNF).

FIG. 12: Toxicity on MCF7 (panel A) or MCF7-mLR (panel B) cells oftargeted modified TNF with individual trimerizing chains coupled to mLRNB (NB C-terminally of TNF).

FIG. 13: Toxicity on MCF7 (panel A) or MCF7-hCD20 (panel B) cells oftargeted modified TNF with individual trimerizing chains coupled tohCD20 NB (NB N-terminally of TNF).

FIG. 14: Comparison of the in vivo toxicity of WT hTNF versus targetedWT and modified sc hTNFs coupled to hCD20 or control Bcll10 NB (NBN-terminally of TNF). (A) Hypothermia (B) Mortality.

FIG. 15: In vivo toxicity of WT or modified (Y86F3x) sc mouse (m)TNFcoupled to control Bcll10 NB (NB N-terminally of TNF). (A) Hypothermia(B) Mortality.

FIG. 16: In vivo anti-tumor effect of WT or modified (Y86F3x) sc mouse(m)TNF coupled to mCD20 or control Bcll10 NB (NB N-terminally of TNF).(A) Tumor growth (B) Mortality.

EXAMPLES Materials and Methods to the Examples

Nanobodies

The nanobody 4-10 directed against the murine leptin receptor (mLR) wasdescribed in Zabeau et al. (2012). Its coding sequence is cloned intothe mammalian expression vector pMET7 (Takebe et al., 1988) in fusionwith the SIgK leader peptide, the HA tag and albumin. Plasmid name:pMET7 SIgK-HA-4.11-Albumin. The anti-Her2 nanobody 1R59B was describedin Vaneycken et al. (2011). The NB 2HCD25 directed against the humanCD20 (hCD20) and the 2MC57 NB against mouse CD20 (mCD20) were generatedusing standard techniques (Gharouhdi et al., 1997; Pardon et al., 2014).The control NB Bcll10 was described in De Groeve et al. (2010).

scTNF

scTNF that consists of three hTNF monomers coupled via GGGGS-linkers(SEQ ID NO: 1) has been described by Boschert et al. (2010). The Y87Qmutation in hTNF was shown to completely abrogate the binding to bothreceptors, TNF-R1 and TNF-R2. Mutating I97 results in reduced binding ofhTNF to both receptors (Loetscher et al., 1993). A whole range ofresidues within hTNF were mutated (QuikChange Site-Directed MutagenesisKit, Stratagene Cat#200518) and tested for their toxic effects on MCF7cells (FIG. 7). We selected the following mutations for the targetedconstructs: Y87Q, Y87F, I97S, I97A, Y115A, Y115G. The coding sequencesof sc hTNF WT-6xGGS, sc hTNF Y87Q3x-6xGGS, sc hTNF I97A3x-6xGGS, sc hTNFY87Q1xI97A2x-6xGGS, sc hTNF Y87Q2xI97A1x-6xGGS, sc hTNF WT1xY87Q2x-6xGGS, sc hTNF WT2x Y87Q1x-6xGGS, sc hTNF I97S3x-6xGGS, sc hTNFY115A3x-6xGGS, sc hTNF Y87F3x, sc hTNF Y115G3x, sc mTNF WT and sc mTNFY86F3x were generated by gene synthesis (GeneArt). The individual chainsare separated by a GGGGS (SEQ ID NO: 1) linker.

scTNF-Nanobody Fusion Construction

The coding sequence of the 1R59B Her2 nanobody was synthesized by PCRfrom the plasmid pHEN6-1R59B with the following primers: forward5′-GTCAAGATCTGGCGGTTCGGCGGCCGCAATGGCCCAGGTGCAGCTGCAG-3′ (SEQ ID NO: 2),reverse 5′-CAGTTCTAGATTACTTATCGTCGTCATCCTTGTAATCCGAACCGCCGTCCGGAGAGGAGACGGTGAC-3′ (SEQ ID NO: 3). This PCR introduces a GGS in between a BgllIand NotI site at the amino terminus and a FLAG tag at the carboxyterminus of the 1R59B nanobody. The PCR product was digested with BgllIand XbaI. The pMK-RQ-sc hTNF WT, pMK-RQ-sc hTNF Y87Q3x and pMK-RQ-schTNF I97A3x were digested with NdeI and BgllI. The digested PCR productand synthetic gene fragments were cloned into NdeI-XbaI digested pMET7SIgK-HA-leptin vector to obtain pMET7 SIgK-HA-sc hTNFWT-6xGGS-1R59B-FLAG, pMET7 SIgK-HA-sc hTNF Y87Q3x-6xGGS-1R59B-FLAG andpMET7 SIgK-HA-sc hTNF I97A3x-6xGGS-1R59B-FLAG. The control vectorswithout the 1R59B nanobody were obtained by inserting the followingannealed oligos containing the GGS and the FLAG tag in between BgllI andXbaI instead of the PCR product: forward:5′GATCTGGCGGTTCGGCGGCCGCAGATTACAAGGATGACGACGATAAGTAAT3′ (SEQ ID NO: 4),reverse: 5′CTAGATTACTTATCGTCGTCATCCTTGTAATCTGCGGCCGCCGAACCGCCA3′ (SEQ IDNO: 5). The control vector with only the 1R59B nanobody was obtained byinserting the following annealed oligos instead of the NdeI-schTNF-BgllI fragment: forward: 5′-TATGATGTGCCCGACTACGCTGGCGGCAGCA-3′ (SEQID NO: 6), reverse 5′-GATCTGCTGCCGCCAGCGTAGTCGGGCACATCA-3′ (SEQ ID NO:7). The length of the GGS linker was adjusted to a GGS linker of 13repeats and 19 repeats by adding 7xGGS or 13xGGS repeats (made by genesynthesis, GeneArt) to the original 6xGGS in between the BgllI and NotIsite.

A similar approach was used to obtain pMET7 SIgK-HA-sc hTNFWT-6x/13x/19xGGS-4.10-FLAG, pMET7 SIgK-HA-sc hTNFY87Q3x-6xGGS-4.10-FLAG, pMET7 SIgK-HA-sc hTNFI97A3x-6x/13x119xGGS-4.10-FLAG, pMET7 SIgK-HA-sc hTNFY87Q1xI97A2x-6x/13x/19xGGS-4.10-FLAG, pMET7 SIgK-HA-sc hTNFY87Q2xI97A1x-6x113x/19xGGS-4.10-FLAG, pMET7 SIgK-HA-sc hTNFWT-6x/13x/19xGGS-2HCD25-FLAG, pMET7 SIgK-HA-sc hTNFI97S3x-6x/13x/19xGGS-2HCD25-FLAG, pMET7 SIgK-HA-sc hTNFI97A3x-6x/13x/19xGGS-2HCD25-FLAG and pMET7 SIgK-HA-sc hTNFY115A3x-6x/13x/19xGGS-2HCD25-FLAG.

To obtain the individual trimerizing hTNF constructs, sc hTNF inpMet7-SIgK-HA-sc hTNF WT-GGS-4.10-Flag was replaced by NdeI-SalI digestof the PCR product obtained with the forward primer5′-CATATGATGTGCCCGACTACGCTGGCGGCAGCAGCTCTAGAACCCCCAGCGATAAGCCT GTG-3′(SEQ ID NO: 8) and the reverse primer 5′-GTCGACCAGGGCAATGATGCCGAAGT-3′(SEQ ID NO: 9) on the plasmids pMet7-SIgK-His-hTNF WT orpMet7-SIgK-His-hTNF I97A. This resulted in the following vectors:pMet7-SIgK-HA-hTNF WT-6xGGS-4.10-Flag and pMet7-SIgK-HA-hTNFI97A-6xGGS-4.10-Flag.

The nanobody-TNF fusion expression constructs with the NB N-terminallyof individual trimerizing or single chain, human or mouse TNF were madein pMet7 and designed as such that each subunit is interchangeablethrough unique restriction sites: AgeI-nanobody-SalI-GGSlinker-NotI-TNF-XhoI-His-XbaI.

pGL3-(IL6-kB)3-fireflyluciferase was kindly provided by W. Vanden Berghe(Vanden Berghe et al., 1998).

Production of the Nanobody-TNF Fusion Proteins for In Vitro Studies

HekT cells were transfected with the protein fusion constructs using thestandard calcium phosphate precipitation method. 48 hours after thetransfection culture mediums were harvested and stored at −20° C. Theconcentration was determined with a commercial hTNF ELISA (DY210, R1Dsystems).

Production of the Nanobody-scTNF Fusion Proteins for In Vivo Studies

FreeStyle™ 293-F cells were transfected with the protein fusionconstructs using the PEIpro™ transfection reagent (PolyPlus,Cat#115-375) according to the manufacturer's guidelines. The endotoxincontent was in all preparations under the detection limit as assessed bya chromogenic Limulus Amebocyte Lysate Assay (Lonza, Cat#50-647U).

Cell Lines

Hek, HekT, Hek-mLR, MCF7, MCF7-hCD20, MCF7-mLR and B16BI6-mCD20 cellswere grown in DMEM supplemented with 10% FCS. The FreeStyle™ 293-F cellline was obtained from Invitrogen, Life Technologies (Cat#R790-07) andmaintained in FreeStyle™ 293 Expression Medium from Gibco, LifeTechnologies (Cat#12338). The human breast cancer SK-BR-3 (ATCC: HTB-30)cell line was obtained from ATCC and maintained in McCoy's 5A mediumsupplemented with 10% FCS.

The Hek-mLR cell line was generated as follows: Flp-ln-293 cells(Invitrogen) were stably co-transfected with a plasmid containing theexpression cassettes for mEcoR and neomycin resistance and with apXP2d2-rPAPI-luci reporter construct (Eyckerman et al. 2001). Stabletransfected clones were isolated in G418 (400 ug/ml)-containing mediumand a clone was selected with high LIF (1 ng/ml)-induced luciferaseactivity. The expression vector pcDNA5/FRT containing the mLR was stablyintegrated in this cell line using the FIp-In recombinase reaction(Invitrogen) and after selection on hygromycin (100 μg/ml) for 10 days.

The human breast cancer MCF7 (ATCC: HTB-22) cell line was obtained fromATCC. The MCF7-hCD20 and MCF7-mLR cell lines were generated as follows:MCF7 cells were stably co-transfected with a plasmid containing theexpression cassette for hCD20 or mLR, and with a plasmid containing theneomycin resistance gene. Stable transfected cells were selected withG418 (1 mg/ml)-containing medium, followed by FACS sorting of hCD20- ormLR-expressing cells.

The B16Bl6-mCD20 cell line was generated as follows: B16Bl6 cells werestably co-transfected with a plasmid containing the expression cassettefor mCD20 and with a plasmid containing the neomycin resistance gene.Stable transfected cells were selected with G418 (2 mg/ml)-containingmedium.

The human breast cancer SK-BR-3 (ATCC: HTB-30) cell line was obtainedfrom ATCC and maintained in McCoy's 5A medium supplemented with 10% FCS.

Measurement of the Luciferase Activities

TNF specific activities were measured by quantifying the luciferaseactivity under the control of the NF-κB promoter. Two days aftertransfection of the NF-κB luciferase reporter(pGL3-(IL6-κB)3-fireflyluciferase) by standard calcium phosphateprecipitation method, cells were stimulated for 6 h with targeted orcontrol sc hTNF. Lysates were prepared (lysis buffer: 25 mM Tris, pH7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100), and35 μl of luciferase substrate buffer (20 mM Tricine, 1.07 mM(MgCO3)4Mg(OH)2.5H2O, 2.67 mM MgSO4.7H2O, 0.1 mM EDTA, 33.3 mMdithiothreitol, 270 μM coenzyme A, 470 μM luciferin, 530 μM ATP, finalpH 7.8) was added per 50 μl of lysate. Light emission was measured for 5s in a TopCount chemiluminescence counter (Packard).

Quantitative RT-PCR

The expression of the TNF inducible gene IL-6 was quantified by RT-PCRrelatively to HPRT in SK-BR-3 cells treated for 6 hours with 500 ng/mlof targeted or control sc hTNF. Total RNA was purified with RNeasycolumns (Qiagen) and equal amounts of RNA (0.5 μg) were used for reversetranscription using the Primescript RT Reagent kit (Takara Bio, Shiga,Japan), following the manufacturer's instructions. The 10-fold dilutedcDNA was added to an RT-QPCR mixture containing 1×SYBR Green I mastermix (04 887 352 001, Roche) and 1 nM gene-specific primers. Assays wereperformed in triplicate on a LightCycler 480 Real-Time PCR Systemthermocycler (Roche Applied Science), and the results were analyzedusing the ΔΔCT method. The following primers were used:

HPRT forward: (SEQ ID NO: 10) 5′TGACACTGGCAAAACAATGCA3′; HPRT reverse:(SEQ ID NO: 11) 5′GGTCCTTTTCACCAGCAAGCT3′; IL-6 forward: (SEQ ID NO: 12)5′GACAGCCACTCACCTCTTCA3′; IL-6 reverse: (SEQ ID NO: 13)5′AGTGCCTCTTTGCTGCTTTC3′.Toxicity Analysis on MCF7 Cells

TNF-specific activities were also measured by assessing the cellulartoxicity on MCF7 cells. 1000 cells were plated in a black 96-well plateand 24 hours later stimulated with the different TNF constructs. After48-72 hours, the number of viable cells was determined using theCellTiter-Glo Luminescent Cell Viability Assay (Promega Cat#G7570)according to the manufacturer's guidelines.

In Vivo Toxicity Analysis

To assess hTNF toxicity in vivo, female 8 weeks old C57BL/6J mice(purchased from Charles River, France) were injected intraperitoneallywith 500 ng rhTNF or sc hTNF-nanobody fusion proteins in combinationwith 10 mg D-Galactosamine (diluted in LPS-free PBS, injected in avolume of 500 μl). Morbidity was monitored by measurement of peripheral(rectal) body temperature. n=2-4 per fusion protein.

To evaluate mTNF toxicity in vivo, mice were injected intravenously with10, 35, 100 or 200 μg sc mTNF-nanobody fusion proteins (injected volume200 μl, dilution in LPS-free PBS). Morbidity was monitored bymeasurement of peripheral (rectal) body temperature. n=2 per dose, perfusion protein, except for 200 μg (n=1).

In Vivo Anti-Tumor Studies

Female C57BL/6J mice of 8 weeks old were shaved and inoculated with6×10⁵ B16Bl6-mCD20 tumor cells subcutaneously in the back (day 0).Treatment was started when the product of the largest perpendiculardiameters was approximately 50 mm² (on day 10). PBS or 35 μg nanobody-scmTNF fusion proteins were administered for 8 consecutive days (day10-17, indicated in FIG. 16A as a grey bar) via paralesional injection(subcutaneous injection near the tumor site but outside the tumornodule). Tumors were measured daily with a caliper and are shown asmean±SEM. Morbidity was monitored by daily measurement of body weightand temperature. n=5 per treatment.

Example 1: The Sc hTNF-Nanobody Fusion Proteins

FIG. 1 shows a schematic representation of the sc hTNF-nanobody fusionproteins either with the nanobody N- or C-terminally of sc hTNF.

Example 2: Targeting TNF Activity on mLR-Expressing Hek Cells

The induction of NF-κB luciferase reporter activity upon TNF stimulationwas tested in HekT cells and in Hek cells that express the murine leptinreceptor (Hek-mLR). As shown in FIG. 2A, WT sc hTNF-induced NF-κBinduction is completely (>1000-fold) or partly (100-fold) abrogated bythe Y87Q3x or the I97A3x mutation, respectively. Moreover, in HekT cellsthat do not express the mLR, all sc hTNF constructs (WT, Y87Q3x andI97A3x) induce similar NF-κB activity independently of the fusion to themLR nanobody (FIG. 2A). In contrast, coupling to the mLR nanobody isable to restore NF-κB induction of sc hTNF I97A3x in Hek cells thatexpress the mLR to a similar extent as WT sc hTNF (FIG. 2B). Weestimated that cells expressing the mLR are 100-fold more sensitive thanparental HekT cells to the nanobody-coupled sc hTNF I97A3x. In contrast,the triple Y87Q mutation did not show any rescue effect of TNFresponsiveness in Hek-mLR cells compared to HekT cells (FIG. 2B).

Example 3: Comparison of Different Mutant Combinations and DifferentLinker Lengths

In order to optimize the constructs, sc hTNF constructs with differentmutations in the individual chains were tested, as well as differentlinker lengths between the sc hTNF and the targeting moiety. The resultsare summarized in FIGS. 3, 4 and 5. sc hTNF I97A3x and sc hTNF Y87Q1xI97A2x do not show activity on Hek cells that do not express the leptinreceptor, but have a clear dose dependent activity when targeted to theleptin receptor.

Example 4: Targeting TNF Activity on her2-Expressing Hek Cells

We generated fusions protein using the α-Her2 nanobody 1R59B and sc hTNFWT, sc hTNF Y87Q3x or sc hTNF I97A3x. The linker between the nanobodyand sc hTNF was either 6xGGS or 19xGGS. These molecules were tested onthe Her2-overexpressing SK-BR-3 breast cancer cell line for theinduction of the IL-6 TNF-inducible gene as determined relatively toHPRT by quantitative RT-PCR.

FIG. 6 shows the fold induction of IL-6 mRNA upon sc hTNF treatment (500ng/ml) compared to IL-6 mRNA levels in untreated cells and cellsstimulated with the Her2 nanobody. In correspondence to thetranscriptional activation of NF-κB, we observe that Y87Q3x mutationcompletely prevents TNF-induced IL-6 production while sc hTNF I97A3x canstill induce IL-6 production but to a lesser extent than WT sc hTNF.When sc hTNF is fused to the nanobody less IL-6 mRNA is produced. Thiscould be due to steric hindrance as the effect is more pronounced withthe 6xGGS linker compared to the longer 19xGGS linker where there islikely more flexibility. By coupling sc hTNF I97A3x to the Her2 nanobodythe induction of IL-6 can be restored to similar levels as WT sc hTNFcoupled to the nanobody through the corresponding linker. In contrast,specific targeting of the more severe Y87Q3x sc hTNF mutant toHer2-expressing cells cannot restore the IL-6 inducing property of schTNF.

Example 5: Comparing the Toxicity of hTNF Mutants on MCF7 Cells

Because of the relatively high residual activity of I97A3x mutant schTNF, we searched for further mutations by measuring the toxicity ofdifferent individual trimerizing hTNF mutants as luciferase activity inMCF7 cells. The activity of the mutants relative to WT individualtrimerizing TNF is shown in FIG. 7. Most mutations do not affect the TNFactivity drastically (>1% of WT) and might be less promising for thedevelopment of targeted constructs because of their possibly remainingsubstantial toxicity. We are more interested in mutations that (almost)completely abrogate TNF function. The use of null mutations (<0.1% ofWT) results in targeted constructs that do not have side effects butthat have as a possible drawback that reactivation upon targeting isless easily accomplished. The mutations that have some residual activity(0.02-5% of WT, particularly 0.1%-1% of WT) have a better chance ofbeing reactivated whilst not being toxic. 6 different mutations coveringan activity range between 0.02 and 5% of individual trimerizing WT TNFwere selected for the development of the targeted modified cytokines:Y87Q (0.02%), I97S and Y115A (0.2%), Y87F (0.5-1%), Y115G (1-2%) andI97A (2-5%).

Example 6: Targeting TNF Activity on mLR-Expressing MCF7 Cells

The toxicity of mLR NB-targeted TNF was assessed on MCF7 and MCF7-mLRcells. Different mutations (I97A3x, I97S3x and Y115A3x) were tested aswell as different linkers between sc hTNF and the mLR NB (6xGGS, 13xGGS,19xGGS). As shown in FIG. 8A, toxicity is reduced 20-fold by the I97A3xmutation and 500-fold by the I97S3x and Y115A3x mutation, which issimilar to what we observed for individual trimerizing TNF (FIG. 7).Moreover, in MCF7 cells that do not express the mLR, fusion to the mLRNB does not alter the activity of WT or mutant sc hTNF (FIG. 8A), whilethis fusion reactivates all sc hTNF mutants on MCF7-mLR cells (FIG. 8B).We estimated that cells expressing the mLR are 100-fold more sensitivethan parental MCF7 cells to the NB-coupled I97S3x and Y115A3x sc hTNF.However, these targeted modified TNFs are still about 20-fold lessactive than WT sc hTNF. In contrast, the I97A3x targeted modified TNF isrestored to WT activity levels on MCF7-mLR cells, which corresponds to a20-fold reactivation (FIG. 8B).

Example 7: Targeting TNF Activity on hCD20-Expressing MCF7 Cells

To assess the effect of other targeting moieties for the targeting ofmodified TNF, we replaced the mLR NB in the constructs of Example 6 withthe hCD20 NB and tested their toxicity on MCF7 cells and MCF7 cells thatexpress hCD20 (MCF7-hCD20). The results are shown in FIG. 9. Asexpected, mLR NB and hCD20 NB targeted modified TNFs behave similarly onparental MCF7 cells (FIGS. 8A & 9A).

Example 8: Targeting TNF Activity on hCD20-Expressing Cells with aDifferent hCD20 NB Fusion Set-Up

We tried to improve the hCD20 NB-TNF constructs by placing the NB infront instead of after sc hTNF. We also tested 2 additional, lessdrastic mutations (Y87F3x and Y115G3x, FIG. 7). The MCF7 and MCF7-hCD20toxicity studies with these constructs are shown in FIG. 10. Sc hTNFcoupled to hCD20 NB exerts the same toxicity on MCF7 cells as thecorresponding mutant coupled to the control Bcll10 NB (FIG. 10A), andthe level of activity is similar as to what we observed for theindividual trimerizing TNF mutants (FIG. 7). This reduced toxicity ofthe mutants is (partially) reverted upon hCD20 targeting on theMCF7-hCD20 cells: hCD20 NB-coupled modified TNF give a 10-fold(Y115G3x), 15-fold (Y87F3x), 100-fold (I97S3x, Y115A3x) or even higher(Y87Q3x) increased activity compared to the corresponding Bcll10 controlNB-coupled sc hTNFs (FIG. 10B). In this experiment, when the hCD20 NB isplaced at the carboxy-terminal end instead of the amino-terminal end ofthe sc hTNF the reactivation is less (FIG. 9B).

Example 9: Comparison of Different Mutant Combinations

Despite the fact that the difference of targeted modified TNF versusnon-targeted modified TNF is at least a 100-fold, some mutations showlower rescued activity than WT activity levels (Y87Q3x) which mightaffect its anti-tumor effects. Alternatively, some mutations still havesome residual activity (I97S3x and Y115A3x) which might lead to some(systemic) toxicity when used in vivo. To overcome these potentialdrawbacks, we tested additional constructs by mutating differentresidues in the individual chains of sc hTNF in order to see whether theactivity levels could thus be further modulated. As shown in FIG. 11,combining different mutations in the single chain can alter the residualactivity on non-targeted cells and the level of reactivation upontargeting.

Example 10: Comparison of Targeted Individual Trimerizing TNF VersusSingle Chain Modified hTNF

To compare the efficiency of targeted individual trimerizing versussingle chain TNF, WT or I97A hTNF was coupled C-terminally to the mLR NBas a monomer. Their toxicity was tested on MCF7 cells and on MCF7 cellsthat express the mLR (MCF7-mLR), and is shown in FIG. 12. Also in theindividual trimerizing form, the I97A mutation is toxic on MCF7 cellsbut to a lesser extent than WT hTNF (FIG. 8A & FIG. 12A). Moreover, whencoupled C-terminally to the mLR nanobody, individual trimerizing—but notsingle chain—TNF becomes less toxic on MCF7 cells, and this is the caseboth for WT and I97A (FIG. 8A & FIG. 12A). Most probably, the 3nanobodies present in the hTNF trimer formed with the individualtrimerizing TNF-mLR NB constructs are sterically hindering the bindingof hTNF to its receptor. This reduced activity can, however, be revertedby targeting to the mLR on MCF7-mLR cells (FIG. 12B). Interestingly,this offers a further level of modulation of activity: one can combinedifferent mutations, as well as use the sterical hindrance to influenceresidual activity and level of reactivation upon targeting.

To address whether this is a general phenomenon, we coupled individualtrimerizing WT and Y115A hTNF N-terminally to Bcll10 or hCD20 nanobodyand tested their toxicity on MCF7 and MCF7-hCD20 cells. As shown in FIG.13A, this coupling does not affect the toxicity of individualtrimerizing WT or Y115A3x hTNF. Moreover, upon targeting, individualtrimerizing Y115A3x hTNF becomes as active as non-targeted individualtrimerizing WT hTNF (FIG. 13B).

Example 11: Assessment of In Vivo Toxicity of Targeted Modified hTNF

To evaluate the toxicity of hTNF mutants preclinically is not evident,since TNF displays a remarkable species specificity in mice. In contrastto mTNF, hTNF only induces lethality at extremely high doses (Brouckaertet al. 1992). Although the reason for this species specificity was longthought to be caused by hTNF not interacting with the murine TNF-R2,pharmacokinetic studies have shown that hTNF is cleared much faster thanmTNF in mice and that the consequential limited hTNF exposure isresponsible for its lack of morbidity (Ameloot et al. 2002).

Nevertheless, when treated with a sensitizing agent such asD-galactosamine, species specificity is abolished and extremely lowdoses (≦500 ng) of hTNF are equally lethal as mTNF (Broeckaert et al.,1992). To assess the in vivo toxicity of the various targeted modifiedhTNFs, we therefore injected mice intraperitoneally with 500 ng ofeither recombinant (r) hTNF, sc hTNF WT or sc mutant hTNF (Y87Q3x orY115A3x). The sc WT and modified hTNF were coupled N-terminally toeither Bcll10 or to hCD20 NB. As shown in FIG. 14, sc WT hTNF is atleast as toxic as rhTNF, causing severe hypothermia and mortality within10 h after injection. Targeted modified hTNF Y87Q3x and Y115A3x did notcause any signs of morbidity (pilo-erection, tremor, lethargy, loss ofgrooming or drop in body temperature; see FIG. 14A for the latter).

Example 12: Assessment of In Vivo Toxicity and Anti-Tumor Effect ofTargeted Modified mTNF

As already mentioned, in vivo toxicity of hTNF cannot be easily studiedin mice. Therefore, as well as because of anticipated anti-tumorexperiments in immunocompetent syngeneic mice, we decided to mutateresidues of mTNF homologous to the ones we selected for hTNF (seeexample 5). As illustrated in FIG. 15, Bcll10NB-sc mTNF WT caused severemorbidity (FIG. 15 A) and 100% mortality (FIG. 15B) when injectedintravenously in doses as low as 10 μg. In contrast, Bcll10NB-sc mTNFY86F3x did not induce mortality (FIG. 15B) nor cause any signs oftoxicity (pilo-erection, tremor, lethargy, loss of grooming or drop inbody temperature; see FIG. 15A for the latter), not even when injectedas an intravenous bolus of 200 μg. Nevertheless, when injected dailyparalesionally in a dose of 35 μg in B16Bl6-mCD20-tumor bearing mice,nanobody-coupled sc mTNF Y86F3x could still reduce/prevent tumor growth,especially when targeted to mCD20 (FIG. 16A). The effect of non-targetedmutant TNF on tumor growth (FIG. 16A) is due to the high dose (35 μg)used, as lower doses more closely mimic PBS-treated animals (data notshown). Daily treatment with the NB-sc mTNF Y86F3x did not cause anysigns of morbidity or mortality, while tumor-bearing mice treated withNB-sc mTNF WT succumbed after 1 or 2 injections (FIG. 16B).

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The invention claimed is:
 1. A method for treating cancer, comprisingadministering an effective amount of a composition comprising a fusionprotein to a patient in need thereof, wherein the fusion proteincomprises: (i) a single chain polypeptide comprising three modifiedhuman TNFs, wherein each modified human TNF comprises a modified aminoacid residue by substitution at the same position selected from Y87,I97, or Y115, and wherein said modified human TNFs have reduced affinitytowards its receptor as compared to wild type human TNF; (ii) a linkersequence; and (iii) a targeting moiety directed to a cellular target,wherein the linker sequence links the modified human TNFs to targetingmoiety, wherein the targeting moiety is directed towards a targetselected from CD20, Her2, c-Met, EGFR, tenascin C, αvβ3 integrin, CD13,CD33, CD47, CD70, Axl, PSCA, and PSMA, and wherein the fusion proteinshows significant biological activity towards cells that are targeted bythe targeting moiety.
 2. The method of claim 1, wherein the Y87 mutationis selected from Y87Q, Y87L, Y87A, or Y87F.
 3. The method of claim 1,wherein the Y115 mutation is selected from Y115A or Y115G.
 4. The methodof claim 1, wherein the I97 mutation is selected from I97A, I97Q orI97S.
 5. The method of claim 1, wherein the linker sequence comprisesGGS.
 6. The method of claim 5, wherein the linker sequence comprises GGSrepeats.
 7. The method of claim 1, wherein the targeting moiety is avariable domain of a camelid heavy chain antibody (VHH).
 8. The methodof claim 1, wherein the targeting moiety is directed towards CD20. 9.The method of claim 1, wherein the targeting moiety is directed towardsHer2.
 10. The method of claim 8, wherein the targeting moiety is a VHH.11. The method of claim 9, wherein the targeting moiety is a VHH. 12.The method of claim 1, wherein Y87 and the targeting moiety is a VHHdirected towards CD20.
 13. The method of claim 12, wherein the mutationis selected from Y87Q, Y87L, Y87A, or Y87F.